Method of hydroxylating 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)-pyridinone by incubation with liver slices

ABSTRACT

Incubation of 3-[2-benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)-pyridinone ##STR1## with a preparation from mammalian organ yields as biotransformation product the 6-hydroxymethyl analog. This product is useful in the prevention or treatment of infection by HIV and the treatment of AIDS.

This is a continuation of application Ser. No. 07/771,594, filed Oct. 4,1991, now abandoned.

This case is related to Merck cases 18122, 18122IA, 18122IB, 18131,18131IA, 18132, 18212, 18212IA, 18317, 18376, 18377 and 18379.

The present invention relates to a novel process for the preparation ofcompound (I) ##STR2## comprising incubation of compound (II), aninhibitor of the reverse transcriptase encoded by human immunodeficiencyvirus (HIV), ##STR3## with a preparation from mammalian organ. Compound(I) or the pharmaceutically acceptable esters thereof inhibit thereverse transcriptase encoded by HIV and is of value in the preventionof infection by HIV, the treatment of infection by HIV and the treatmentof the resulting acquired immune deficiency syndrome (AIDS).

BACKGROUND OF THE INVENTION

A retrovirus designated human immunodeficiency virus (HIV) is theetiological agent of the complex disease that includes progressivedestruction of the immune system (acquired immune deficiency syndrome;AIDS) and degeneration of the central and peripheral nervous system.This virus was previously known as LAV, HTLV-III, or ARV. A commonfeature of retrovirus replication is reverse transcription of the RNAgenome by a virally encoded reverse transcriptase to generate DNA copiesof HIV sequences, a required step in viral replication. It is known thatsome compounds are reverse transcriptase inhibitors and are effectiveagents in the treatment of AIDS and similar diseases, e.g.,azidothymidine or AZT.

Nucleotide sequencing of HIV shows the presence of a pol gene in oneopen reading frame [Ratner, L. et al., Nature, 313, 277(1985)]. Aminoacid sequence homology provides evidence that the pol sequence encodesreverse transcriptase, an endonuclease and an HIV protease [Toh, H. etal., EMBO J. 4, 1267 (1985); Power, M.D. et al., Science, 231, 1567(1986); Pearl, L.H. et al., Nature 329, 351 (1987)].

The compound prepared by the process of this invention is an inhibitorof HIV reverse transcriptase. Since the compound itself is a metabolite,it is better adapted as a pharmaceutical product. Further, the compoundof the present invention does not require bio-activation to beeffective.

BRIEF DESCRIPTION OF THE INVENTION

The novel process of this invention comprises incubation of Compound II##STR4## with a preparation from a mammalian organ, and isolation of theresulting biotransformation product, Compound (I), in a conventionalmanner: ##STR5## This compound is useful in the inhibition of HIVreverse transcriptase, the prevention of infection by HIV, the treatmentof infection by HIV and in the treatment of AIDS and/or ARC, either as acompound, pharmaceutically acceptable salt (when appropriate), hydrate,ester, pharmaceutical composition ingredient, whether or not incombination with other antivirals, anti-infectives, immunomodulators,antibiotics or vaccines.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The novel process of this invention comprises incubation of compound(II) ##STR6## with a preparation from mammalian organ, and isolation ofthe resulting biotransformation product, compound (I), in a conventionalmanner: ##STR7## 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-hydroxymethyl-2(1H)-pyridinone.

In general, compound (I), which is a 6-hydroxymethyl oxidation product,can be produced by incubating an appropriate amount of substratecompound (II) with certain mammalian tissues or cell cultures in anaqueous medium suitable for enhancing the viability of the tissues orcells. Metabolite (I) may be produced by incubation of compound (II)with a preparation from mammalian organ containing: a) preparations fromsurgically derived specimens including liver, kidneys, lungs and skin,both from animals and human beings; b) prenatal and gestational tissues;c) cell cultures; d) subcellular fractions like microsomes, S9 andcytosol; and/or e) purified mixed function oxidases. These metabolitescould also be formed in vivo in animals and human beings. The preferredtissue for production of compound (I) is liver, especially rat liverslices.

The appropriate amount of tissue or cell culture to be used with a givenamount of substrate compound will vary with the particular type ofculture used. An appropriate ratio of substrate compound (II) to beincubated with liver tissue (mg:g, wet weight) ranges from about 1:0.3to 1:3.0, preferably 1:1.5. When using surgically derived specimens,especially liver, the specimen is preferably cut into slices withthickness ranging from about 100μ to 1000μ, and more preferably fromabout 250 to 400μ.

Aqueous media sufficient in amount and kind to keep the tissue or cellshealthy in the incubation process should be used. These media are knownand available in the art of drug metabolism and include various buffersand standard culture media with or without additives. A few examples ofvarious culture media that may be employed are Williams' Medium E,Waymouth's Medium, Dulbecco's Medium, RPMI Medium and the like. Culturemedia can be replaced by general buffers such as phosphate buffers.Various additives that may be used to enhance the viable life of thecells and tissues are a) serum from bovine, horse, chicken, goat, sheep,rabbit and the like; b) HEPES or MOPS; c) gentamycin; and d) insulin,for example. A preferred medium for incubation of substrate compound(II) with rat liver slices is Williams' Medium E.

The material is incubated at a temperature between 35° and 39° C.,preferably 37° C., and at a pH between 7.2 and 7.6, preferably 7.4,under an atmosphere of 0% to 5% carbon dioxide in oxygen, or air. Thematerial is incubated for a period of time necessary to complete theoxidative biotransformation as monitored by HPLC (high performanceliquid chromatography), usually for a period of about four hours whenincubated with rat liver slices.

The biotransformation product (I) can be isolated and purified from theincubation mixture by extraction with a conventional solvent, such asmethylene chloride, ethyl acetate, acetonitrile, methanol and the like,pH adjustment, treatment with a conventional resin (e.g. anion or cationexchange resin, non-ionic adsorption resin, etc.), treatment with aconventional adsorbent (e.g. activated charcoal, silicic acid, silicagel, cellulose, alumina, etc.), crystallization, recrystallization, andthe like. A preferred recovery method is solvent extraction,particularly using ethyl acetate. A preferred purification methodinvolves the use of chromatography, especially HPLC, using a bondedsilica gel column. Eluant mixtures for chromatography can be composed ofwater and an organic solvent such as methanol, acetonitrile and thelike, and may optionally include a small amount of base, such asammonium bicarbonate, or an acid, such as trifluroacetic acid (TFA) orphosphoric acid. A preferred eluant is composed of acetonitrile andwater containing 0.1% ammonium bicarbonate and is run through the columnwith a linear gradient.

A process for making esters of (I) is also encompassed by the presentinvention. Such esters are those which would readily occur to theskilled artisan, and include, for example, C₁₋₄ alkyl esters.

The compound of the present inventions is useful in the inhibition ofHIV reverse transcriptase, the prevention or treatment of infection bythe human immunodeficiency virus (HIV) and the treatment of consequentpathological conditions such as AIDS. Treating AIDS or preventing ortreating infection by HIV is defined as including, but not limited to,treating a wide range of states of HIV infection: AIDS, ARC (AIDSrelated complex), both symptomatic and asymptomatic, and actual orpotential exposure to HIV. For example, the compound of this inventionis useful in treating infection by HIV after suspected past exposure toHIV by, e.g., blood transfusion, organ transplant, exchange of bodilyfluids, bites, accidental needle stick, or exposure to patient bloodduring surgery.

EXAMPLE 13-[2-(Benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)-pyridinone,substrate compound (II)

Step A: Preparation of 3-cyano-5-ethyl-6-methyl-2-(1H)-pyridinone

According to the method described in J. Heterocyclic Chem., 24, 351(1987), a mixture of 2-ethyl-3-oxobutanal, sodium salt (37.5 g, 0.275mmol), cyanoacetamide (25.2 g, 0.30 mol), aqueous piperidinium acetate(22 mL) [prepared from glacial acetic acid (4.2 mL), water (10 mL) andpiperidine (7.2 mL)] in water (775 ml) was refluxed for four hours.Glacial acetic acid (30 ml) was added cautiously (much foaming) as theproduct precipitated. Upon cooling to room temperature, the product wascollected by filtration, washed with cold water and air dried to yield22.3 g (50%), m.p. 237°-240° C.

Step B: Preparation of 5-ethyl-6-methyl-2-(1H)-pyridinone-3-carboxylicacid

An initial suspension of 5-ethyl-6-methyl-2-(1H)-pyridinone (4.86 g, 30mmol) in 6N HCl (100 mL) was heated at reflux for twenty hours. Uponcooling, the product crystallized and was collected by filtration,washed with cold water and air dried to yield 3.73 g (69%).

Step C: Preparation of methyl 2-chloro-5-ethyl-6-methyl nicotinate

A mixture of 5-ethyl-6-methyl-2-(1H)-pyridinone-3-carboxylic acid (3.62g, 20 mmol) and phosphorus pentachloride (4.38 g, 21 mmol) was heated,under a nitrogen atmosphere, at 100°-120° C. for 1.5 hours. The cooledresidue was diluted with chloroform (70 mL) and then methanol (15 mL)was added. After stirring for 2-16 hours, the solution was poured intoice/water. The organic layer was separated and washed successively withwater, saturated aqueous NaHCO₃, dried (Na₂ SO₄), filtered and thesolvent evaporated. This dark amber oil was dissolved in hexane,filtered through a pad of charcoal and the solvent evaporated to yield3.31 g (78%) of pure product as a light yellow oil.

Step D: Preparation of methyl 2-methoxy-5-ethyl-6-methylnicotinate

To a solution of sodium metal (0.55 g, 24 mmol) dissolved in anhydrousmethanol (15 mL), under a nitrogen atmosphere, was added a solution ofmethyl 2-chloro-5-ethyl-6-methylnicotinate (3.18 g, 14.9 mmol) in drymethanol (5 mL). This solution was refluxed and monitored by tlc (thinlayer chromatogram) until the starting material had been consumed (about24 hours). The cooled mixture was diluted with diethyl ether (50 mL),washed with water, saturated aqueous NaHCO₃, dried (Na₂ SO₄), filteredand the solvent evaporated to yield 2.28 g (73%) of pure product as alight yellow oil.

Step E: Preparation of 2-methoxy-3-hydroxymethyl-5-ethyl-6-methylpyridine

To a solution of methyl 2-methoxy-5-ethyl-6-methylnicotinate (2.28 g,10.9 mmol) in anhydrous tetrahydrofuran (50 mL), under a nitrogenatmosphere, was added cautiously lithium aluminum hydride (0.77 g, 20mmol). After refluxing this mixture for 15-20 hours, saturated aqueousNa₂ SO₄ was added carefully to quench the cooled reaction mixture. Thismixture was diluted with more THF, dried (Na₂ SO₄), filtered and thesolvent evaporated. This residue was chased with ethanol/toluene toremove traces of water and triturated with hexane as the product slowlycrystallized out to give 1.30 g (66%), mp 53°-55° C.

Step F: Preparation of 2-methoxy-5-ethyl-6-methyl nicotinaldehyde

Activated manganese dioxide (2.0 g) was added to a solution of2-methoxy-3-hydroxymethyl-5-ethyl-6-methylpyridine (1.18 g, 6.5 mmol) indry benzene (20 mL) and refluxed 5-10 hours. The warm suspension wasfiltered through a pad of anhydrous Na₂ SO₄ and evaporated to yield 1.05g (90%) of a viscous oil which solidified.

Step G: Preparation of2-[2(R/S)-hydroxy-2-(2-methoxy-5-ethyl-6-methyl-pyridin-3-yl)ethyl]benzoxazole

To a solution of 2-methylbenzoxazole (266 mg, 1.7 mmol) in anhydrous THF(4 mL), cooled to -100° C. under an argon atmosphere, was added 1.6Mn-butyllithium/hexane (1.05 mL) slowly over 35 minutes. After 0.5 hour asolution of 2-methoxy-5-ethyl-6-methylnicotinaldehyde (300 mg, 1.7 mmol)in dry THF (1 mL) was added dropwise. The reaction was allowed to warmto room temperature and poured onto crushed ice. This mixture wasextracted with diethyl ether. The combined extracts were dried (MgSO₄)and the solvent removed to give an oil which was flash chromatographedover silica gel. Elution with ethyl acetate/hexane (1:19) gave 340 mg(65%) of analytically pure racemic product, mp 102°-103° C.

Anal. Calcd for C₁₈ H₂₀ N₂ O₃.0.1 H₂ O: C, 68.81; H, 6.48; N, 8.92.Found: C, 68.80; H, 6.76; N, 8.95.

Step H: Preparation of3-[2-(benzoxazol-2-yl)ethenyl]-5-ethyl-6-methyl-2-(1H)-pyridinone

A mixture of2-[2(R/S)-hydroxy-2-(2-methoxy-5-ethyl-6-methylpyridin-3-yl)ethyl]benzoxazole(72 mg, 0.23 mmol) and pyridine hydrochloride (133 mg, 1.2 mmol), undera nitrogen atmosphere, was placed in a preheated oil bath (165° C.) for5 minutes. The reaction flask was removed, cooled, and water added togive a solid. This crude product was extracted into chloroform, dried(MgSO₄) and the solvent evaporated to yield 49 mg (75%) of pure product.Recrystallization from methanol gave 15 mg of analytically pure product,mp 262°-264° C.

Anal. Calcd for C₁₇ H₁₆ N₂ O₂ : C, 72.83; H, 5.75; N, 10.00. Found: C,72.93; H, 5.95; N, 9.99.

Step I: Preparation of3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)-pyridinone

A solution of 80% pure3-[2-(benzoxazol-2-yl)ethenyl]-5-ethyl-6-methyl-2-(1H)-pyridinone 200mg) in methanol/ethanol/THF (25 mL, 1:1:1) was hydrogenated atatmospheric pressure over 5% palladium/charcoal for four hours. Afterfiltering off the catalyst, the solvents were evaporated and the residueflash chromatographed over silica gel. Elution with 2% methanol-98%chloroform gave 75 mg of analytically pure product, mp 155°-156.5° C.

Anal. Calcd. for C₁₇ H₁₈ N₂ O₂ C, 72.31; H, 6.43; N, 9.92. Found: C,72.45; H, 6.52; N, 9.99.

EXAMPLE 2

Another, simple procedure for the synthesis of the product of Example 1is as follows.

Step A: Preparation of 3-cyano-5-ethyl-6-methyl-2-(1H)-pyridinone

Accordingly to the method described in J. Heterocyclic Chem., 24, 351(1987), a mixture 2-ethyl-3-oxobutanol, sodium salt (37.5 g, 0.275 mol),cyanoacetamide (25.2 g, 0.30 mol), aqueous piperidinium acetate (22 mL)[prepared from glacial acetic acid (4.2 mL), water (10 mL) andpiperidine (7.2 mL)] in water (775 ml) was refluxed for four hours.Glacial acetic acid (30 ml) was added cautiously (much foaming) as theproduct precipitated. Upon cooling to room temperature, the product wascollected by filtration, washed with cold water and air dried to yield22.3 g (50%), m.p. 237°-240° C.

New Step B: Preparation of 2-chloro-3-cyano-5-ethyl-6-methylpyridine

3-Cyano-5-ethyl-6-methyl-2-(1H)-pyridinone (22.9 g, 0.141 mol) andphosphorus pentachloride (33.1 g, 0.159 mol) were intimately mixed andheated at 110°-120° C. for one hour. The liquified solids were pouredonto crushed ice and water and the semi-solid was extracted intochloroform. This extract was washed with water, saturated aqueousNaHCO₃, dried (Na₂ SO₄), filtered and evaporated. This amber oil wasdissolved in hexane and the insoluble material was removed when filteredthrough a pad of charcoal. Removal of the solvent gave a light yellowoil which solidified (17.7 g). Trituration of this solid with coldhexane yielded 15.6 g (61%) of pure product, m.p. 63°-64° C.

New Step C: Preparation of 2-methoxy-3-cyano-5-ethyl-6-methylpyridine

Sodium metal (3.25 g, 0.141 mol) was dissolved in dry methanol (100 mL)under a nitrogen atmosphere. When solution was complete, a slurry of2-chloro-5-ethyl-6-methylpyridine (17.95 g, 99.4 mmol) in dry methanol(70 mL) was added and the reaction was warmed at 60° C. for 15-20 hours.After cooling the reaction mixture, diethyl ether (250 mL) and water(200 mL) were added. The ether layer was separated and washed withwater, dried (Na₂ SO₄), filtered and evaporated to give a light yellowsolid (17.5 g). This solid was triturated with cold hexane to yield 14.4g (82%) of pure product, m.p. 59°-61° C.

New Step D: Preparation of 2-methoxy-5-ethyl-6-methylnicotinaldehyde

To a solution of 2-methoxy-3-cyano-5-ethyl-6-methylpyridine (1.0 g, 5.68mmol) in dry tetrahydrofuran (50 mL) under a nitrogen atmosphere andcooled to -70° C., was added 1.3M diisobutyl aluminum hydride/THF (17.4mL, 22.7 mmol). The resulting mixture was allowed to warm to roomtemperature and stir for 15-20 hours. The reaction mixture was acidifiedwith 1N hydrochloric acid and then neutralized with aqueous sodiumbicarbonate. Water was then added and the product extracted into diethylether. The etheral extract was dried (Na₂ SO₄), filtered and the solventevaporated. This residue was flash chromatographed on silica gel elutingwith 10% diethyl ether/pentane to give 610 mg (61%) of product.

Step E (old step G): Preparation of2-[2(R/S)hydroxy-2-(2-methoxy-5-ethyl-6-methylpyridin-3-yl)ethyl]-benzoxazole

To a solution of 2-methylbenzoxazole (226 mg, 1.7 mmol) in anhydrous THF(4 mL), cooled to -100° C. under an arton atmosphere, was added 1.6Mn-butyllithium/hexane (1.05 mL) slowly over 35 minutes. After 0.5 hour asolution of 2-methoxy-5-ethyl-6-methylnicotinaldehyde (300 mg, 1.7 mmol)in dry THF (1 mL) was added dropwise. The reaction was allowed to warmto room temperature and poured onto crushed ice. This mixture wasextracted with diethyl ether. The combined extracts were dried (MgSO₄)and the solvent removed to give an oil which was flash chromatographedover silica gel. Elution with ethyl acetate/hexane (1:9) gave 340 mg(65%) of analytically pure racemic product, mp 102°-103° C.

Anal. Calcd for C₁₈ H₂₀ N₂ O₃.0.1 H₂ O: C, 68.81; H, 6.48; N, 8.92.Found: C, 68.80; H, 6.76; N, 8.95.

Step F (old step H): Preparation of3-[2-(benzoxazol-2-yl)-ethenyl]-5-ethyl-6-methyl-2-(1H)pyridinone

A mixture of2-[2(R/S)-hydroxy-2-(2-methox-5-ethyl-6-methylpyridin-3-yl)ethyl]benzoxazole(72 mg, 0.23 mmol) and pyridine hydrochloride (133 mg, 1.2 mmol), undera nitrogen atmosphere, was placed in a preheated oil bath (165° C.) for5 minutes. The reaction flask was removed, cooled, and water added togive a solid. This crude product was extracted into chloroform, dried(MgSO₄) and the solvent evaporated to yield 49 mg (75%) of pure product.Recrystallization from methanol gave 15 mg of analytically pure product,mp 262°-264° C.

Anal. Calcd for C₁₇ H₁₆ N₂ O₂ : C, 72.83; H, 5.75; N, 10.00. Found: C,72.93; H, 5.95; N, 9.99.

Step G (old step I): Preparation of3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)pyridinone

A solution of 80% pure3-[2-(benzoxazol-2-yl)ethenyl]-5-ethyl-6-methyl-2-(1H)-pyridinone (200mg) in methanol/ethanol/THF (25 mL, 1:1:1) was hydrogenated atatmospheric pressure over 5% palladium/charcoal for four hours. Afterfiltering off the catalyst, the solvents were evaporated and the residueflash chromatographed over silica gel. Elution with 2% methanol-98%chloroform gave 75 mg of analytically pure product, mp 155°-156.5° C.

Anal. Calcd, for C₁₇ H₁₈ N₂ O₂ C, 72.31; H, 6.43; N, 9.92. Found: C,72.45; H, 6.52; N, 9.99.

EXAMPLE 3 Incubation With Rat Liver Slices.

Incubation of tritiated3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)-pyridinone (parentcompound, 14.18 μmol) with rat liver slices (6.1 g) in 100 ml ofWilliams Medium E at pH 7.4 and at 37° C. under a partial atmosphere of95% O₂ +5% CO₂ resulted in ˜32% metabolism in four hours. Ethyl acetateextraction gave a recovery of ˜70% of the tritiated material, while ˜25%(˜9 HPLC peaks) remained in the aqueous residue. Analysis of the aqueousresidue showed that the unreacted parent compound was quantitativelyextracted in ethyl acetate. HPLC of the organic extract on a μBondapakC₁₈ (7.8×300 mm) column eluted with 0.1% TFA containing MeCN-H₂ Ogradient showed three significant radioactive peaks: M1 (˜37% of thetotal metabolites), M2 (15%) and M3 (6%). The peaks M1 and M3 had UVspectra similar to the parent compound (λ_(max) ˜233, 270, 276, 313 nm),while M2 was lacking the UV band around 313 nm, suggesting a disruptionin the pyridone conjugation system of M2. Notably, M1 and M2 co-elutedunder analytical conditions using a (3.9×300 mm) C₁₈ column.

The metabolite, M3, was separated on the prep HPLC column using a 0.1%TFA based acetonitrile-water gradient, then repurified using amethanol-water gradient.

EXAMPLE 4 STRUCTURAL DETERMINATION: NMR

The structure of the M3 liver-slice metabolite of the parent compoundwas determined by NMR spectroscopy (CD₃ CN, 500 MHz) to be thehydroxylated derivative shown below. ##STR8##

The 1D proton NMR spectrum of I (prepared from rat liver slices)dissolved in CD₃ CN is similar to the spectrum of II with the execptionthat the 6-methyl group resonance is replaced with a resonance at 4.45ppm. See Table A. This chemical shift of the new resonance isappropriate for a methylene between a hydroxyl group and an unsaturatedring as shown in the M3 structure. The slight perturbation of nearbyproton chemical shifts are in agreement with the 6-methyl being the siteof metabolic hydroxylation. The integral of the 4.45-ppm resonance isslightly greater than 2 which indicates that it overlaps anotherresonance, possibly the OH resonance. An expansion of the 4.45-ppmresonance reveals a shoulder peak.

                  TABLE A                                                         ______________________________________                                                      II        I in                                                  Proton        in CD.sub.2 CN                                                                          CD.sub.3 CN                                           ______________________________________                                        1             9.40      9.27                                                  4             7.13      7.18                                                  7             2.93      2.96                                                  8             3.18      3.20                                                  9             2.27      2.25                                                  10            0.94      0.95                                                  11            2.14      4.45                                                   4'           7.62      7.62                                                   5'           ˜7.31                                                                             ˜7.32                                            6'           ˜7.31                                                                             ˜7.32                                            7'           7.54      7.54                                                  ______________________________________                                    

Key assignments of the benzoxazole ring protons were achieved bycorrespondence with the spectrum of 2-methylbenzoxazole (Aldrich; seebelow for full assignment by carbon and proton NMR studies).

    ______________________________________                                        2-Methylbenzoxazole: (ca. 100 mg/ml in CD.sub.3 CN; 500 MHz;                  assignments via short and long-range HETCOR experiments)                       ##STR9##                                                                     position    proton δ (ppm)                                                                      carbon δ (ppm)                                  ______________________________________                                        2           --          165.1                                                 2-CH.sub.3  2.51         14.5                                                 3a          --          142.7                                                 4           7.61        120.1                                                 5           ˜7.30 125.0                                                 6           ˜7.30 125.3                                                 7           7.52        111.1                                                 7a          --          151.9                                                 ______________________________________                                    

EXAMPLE 5 STRUCTURAL DETERMINATION: FAB/MS

Fab-Mass spectral analysis of I in glycerol matrix showed (M+H)⁺ peak atm/z 299, consistent with hydroxylation of the parent or substratecompound II.

EXAMPLE 6 REVERSE TRANSCRIPTASE ASSAY

The assay measures the incorporation of tritiated deoxyguanosinemonophosphate by recombinant HIV reverse transcriptase (HIV RT_(R)) (orother RT) into acid-precipitable cDNA at the Km values of dGTP and polyr(C).oligo d(G)₁₂₋₁₈. The inhibitor of the present invention inhibitsthis incorporation.

Thirty uL of a reaction mixture containing equal volumes of: 500 mMTris.HCl (pH 8.2), 300 mM MgCl₂, 1200 mM KCl, 10 mM DTT, 400 μg/mL polyr(c).oligo d(G) [prepared by dissolving 1.5 mg (25 U) poly r(C).oligod(G) in 1.5 ml sterile distilled H₂ O and diluting to 400 μg/ml], 0.1μCi/μl [³ H] dGTP, 160 μM dGTP, were added to 10 μl sterile distilled H₂O, and 2.5 μl of potential inhibitor. An aliquot of 10 μL of 5 nMpurified HIV RT_(R) was added to initiate the reaction. The mixture wasincubated at 37° C. for 45 minutes.

After incubation is complete, the tubes were cooled in ice for 5minutes. Ice-cold 13% TCA containing 10 mM NaPP_(i) (200 μl) are addedand the mixture incubated on ice for 30 minutes. The precipitated cDNAis removed by filtration using presoaked glass filters [TCA, NaPP_(i) ].The precipitate is then washed with 1N HCl, 10 mM NaPP_(i).

The filter discs are then counted in a scintillation counter.

Under these conditions [dGTP] and poly r(C).oligo d(G)₁₂₋₁₈ each areapproximately equal to the appropriate Km value. Approximately 5-6,000cpm of [³ H] GMP are incorporated into acid-precipitable material. TheRT reaction is concentration- and time-dependent. DMSO (up to 5%) doesnot affect enzyme activity. The calculated IC₅₀ value for the compound(I) of this invention is about 44 nM.

EXAMPLE 73-[2-(Benzoxazol-2-yl)ethyl]-5-ethyl-6-hydroxymethylpyridin-2(1H)-one

Step 1: Prep of 4-Benzyloxy-3-oxo-2-ethylbutanal

A solution of 90% pure 1(N-morpholino)-1-butene (9.3 gm, 60 mmol) andtriethylamine (8.4 mL, 60 mmol) in tetrahydrofuran (85 mL), under anitrogen atmosphere, was warmed to 70° C. and benzyloxyacetyl chloride(9.45 mL, 60 mmol) was added dropwise via a syringe. The cloudy yellowsolution was warmed for 45 minutes and then cooled to room temperature.An aqueous solution of 10% HCl (75 mL) was added and the two phasemixture was stirred for 1 hour. This mixture was partitioned intomethylene chloride and the organic layer was separated, dried (Na₂ SO₄)filtered and the solvent evaporated to give a yellow oil (14.7 g) whichcontained 80% desired product. This oil was used as is.

Step 2: Prep. of 3-Cyano-5-ethyl-6-benzyloxymethylpyridin-2(1H)-one

To a solution of crude 4-benzyloxy-3-oxo-ethylbutanal (6.4 gm, 24 mmol)and malononitrile (2.22 g, 33 mmol) in ethanol (30 mL) was added aceticacid (2.0 mL, 35 mmol), followed by dropwise addition of piperidine(2.37 mL, 24 mmol) to give a dark reddish brown solution. After stirringfor 14 hours a copious precipitate formed. This mixture was warmed at70° C. for 15 hours and then allowed to cool to room temperature. Thisblackish mixture was diluted with ethanol and the precipitated productwas filtered, rinsed with ethanol and diethyl ether to give off-whiteproduct (1.46 g), mp: 141°-143° C. Additional material was obtained byevaporation of filtrate. Extraction of the residue into choroform wasconducted, and after washing this extract with saturated aqueous NaHCO₃,filtration through a pad of charcoal was performed. This amber solutionwas evaporated and the residue triturated with diethyl ether to giveadditional product (0.47 g). The estimated yield of this reaction was30%.

Step 3: Prep. of 2-Benzyloxy-3-cyano-5-ethyl-6-benzyloxymethylpyridine

To a partial suspension of3-cyano-5-ethyl-6-benzyloxymethylpyridin-2(1H)-one (1.36 g, 5.08 mmol)in dry benzene (20 mL) was added benzyl bromide (0.80 mL, 6.7 mmol) andthen silver carbonate (1.43 g, 5.2 mmol). This mixture was covered withaluminum foil and allowed to stir at room temperature. After 24 hours,the reaction was estimated to be ˜80% complete. Additional silvercarbonate (0.40 g, 1.45 mmol) was added and the mixture was stirred foranother 24 hours until complete. The silver salts were removed byfiltration, rinsed with benzene and the combined benzene washings wereevaporated to give pure product as an oil (2.09 g, quantitative).

Step 4: Prep. of 2-benzyloxy-5-ethyl-6benzyloxymethyl nictinaldehyde

To a solution of 2-benzyloxy-3-cyano-5-ethyl-6-benzyloxymethyl-pyridine(2.57 g, 6.2 mmol) in dry toluene (10 mL), under a nitrogen atmospherein an ice/acetone bath, was added dropwise a solution of 1.5Mdiisobutylaluminum hydride/toluene (4.6 mL, 6.9 mmol). After stirring atroom temperature for 15 hours, the reaction was cautiously poured into10% HCl (30 mL), stirred for 0.5 hours and the product extracted intodiethyl ether. The ethereal solution was dried, filtered thru a pad ofcharcoal and evaporated to give a pale yellow oil (1.71 g). This oil wasfurther purified by passing a benzene solution through a plug of silicagel to give pure product (1.44 g, 55% yield) upon evaporation.

Step 5: Prep of3-[2-(Benzoxazol-2-yl)ethenyl]-5-ethyl-6-benzyloxymethyl-2-benzyloxypyridine

To a suspension of [(benzoxazol-2-yl)methyl]-triphenylphosphoniumchloride (1.77 g, 4.12 mmol) in dry tetrahydrofuran (20 mL), under anitrogen atmosphere, was added 60% NaH/mineral oil (0.42 g, 10 mmol).After 0.5 hour a solution of2-benzyloxy-5-ethyl-6-benzyloxymethylnicotinaldehyde (1.44 g, 4.0 mmol)in tetrahydrofuran (10 mL) was added and the reaction mixture wasrefluxed for 23 hours. The reaction was cooled, neutralized with aceticacid and partitioned between water and chloroform. The chloroform layerwas washed with NaHCO₃ solution, dried, filtered through a pad ofcharcoal and evaporated. The material was dissolved in chloroform andflash chromatographed through silica gel to give a pure product as ayellow oil (1.40 g, 74% yield). The product was a mixture of cis andtrans olefins (ratio ˜0.37).

Step 6: Prep of3-[2-(Benzoxazol-2-yl)ethyl]-5-ethyl-6-benzyloxymethylpyridine-2-(1H)-one

A solution of cis/trans3-[2-(benzoxazol-2-yl)ethenyl]-5-ethyl-6-benzyloxymethyl-2-benzyloxypyridine(1.40 g, 2.94 mmol) in dry tetrahydrofuran (20 mL) and methanol (75 mL)containing 10% Pd on charcoal (215 mg) as a catalyst was hydrogenated atatmosphere pressure for 20 hours. The catalyst was filtered off and thesolution evaporated to give a white residue. This residue was trituratedwith diethyl ether to give pure product (0.78 gm, 69% yield), mp:140°-142° C.

Step 7: Prep of3-[2-(Benzoxazol-2-yl)ethyl]-5-ethyl-6-hydroxymethylpyridine-2-(1H)-one

A solution of3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-benzyloxymethylpyridin-2-(1H)-one(198 mg, 0.51 mmol) in dry methylene chloride (6 mL) was cooled in anice/acetone bath and 1M boron tribromide hexane (1.5 mL, 1.5 mmol) wasadded dropwise to give a white precipitate. The suspension was stirredfor one hour and then the reaction was quenched by addition of saturatedaqueous NaHCO₃ (10 mL). After stirring for 0.5 hour, the product wasextracted into CH₂ Cl₂, dried, filtered and the solvent evaporated togive a solid. Trituration with diethyl ether gave product as a tan solid(138 mg, 90% yield). Recrystallization from ethyl acetate afforded alight yellow solid (100 mg), mp: 155°-156° C.

Anal. Calcd for C₁₇ H₁₈ N₂ O₃ : C, 68.44; H, 6.08; N, 9.39. Found: C,68.15; H, 6.03; N, 9.07.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention emcompasses all ofthe usual variations, adaptations, or modifications, as come within thescope of the following claims and its equivalents.

What is claimed is:
 1. A method of preparing the compound ##STR10## or apharmaceutically acceptable ester thereof, comprising the steps of (1)providing a quantity of3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1H)pyridinone,(2)incubating the compound of step 1 with rat liver slices, and (3)isolating the compound.